Jumat, 21 Juni 2019

Effects of Bacillus amyloliquefaciens as a probiotic strain on growth performance, cecal microflora, and fecal noxious gas emissions of broiler chickens

Jual Culture Bacillus amyloliquifaciens
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This experiment was conducted to investigate the effects of Bacillus amyloliquefaciens probiotic (BAP) as a direct-fed microbial on growth performance, cecal microflora, serum immunoglobulin levels, and fecal noxious gas emissions of broiler chickens. A total of 400 one-day-old broiler chicks (Ross 308) were randomly assigned to 1 of 5 treatment diets formulated to supply 0, 1, 5, 10, and 20 g/kg of BAP and were fed for 35 d. Each treatment had 8 replicate pens with 10 birds per replicate. On completion of the growth trial, fecal samples were collected, and ammonia (NH3) and hydrogen sulfide (H2S) emissions were measured. Increasing concentration of BAP had positive linear effect on the ADG of broilers (P < 0.05) throughout the experimental period, with the highest values being observed in broilers offered 20 g/kg of BAP. The ADFI increased linearly (P < 0.02) with the inclusion of BAP during the overall experimental period (d 0 to 35). Providing BAP had a negative linear effect on FCR from d 0 to 21 and d 0 to 35 (P < 0.01). Supplementation with BAP did not affect cecal Lactobacillus and Bacillus content, but exerted negative linear effect on cecal Escherichia coli (P < 0.05) with increasing the level of BAP in broiler diets. Additionally, BAP modified immune response of broilers by linearly increasing serum IgG and IgA (P < 0.01). Dietary BAP resulted in decreased fecal NH3 emissions at 0 (linear, P < 0.001), 3, 6, 12, 24, and 48 h of incubation (linear, P < 0.05; quadratic, P < 0.01). Supplementation of BAP exerted negative linear and quadratic effects on fecal emissions of H2S (P < 0.001) throughout the incubation period except at 48 h, and the optimum effect was found when BAP was provided at 5 g/kg of diet. Based on these results, Bacillus amyloliquefaciens could be suggested as a potential feed additive of broiler diets.

Issue Section:
During the last few decades, livestock production has evolved considerably from largely integrated farming to intensive systems of rearing. Although intensified livestock production is economically effective, it has led to an increase in stress that animals are subjected to, which has resulted in decreased immune function and productivity, overuse or misuse of antibiotics to improve immunity and growth performance, and public complaints concerning odor emissions, and so on. According to the American Meat Institute (International Post, 2013), there was a 0.2% increase in meat and poultry production in 2011 compared with 2010, whereas antibiotics use increased by 2%, meaning that the average amount of antibiotics used to produce 1 kg of meat increased over that time span. The use of antibiotics has been further compounded by broiler diet formulations, which commonly contain high concentrations of trace minerals and may have deleterious effects on the bacteria needed for effective nutrient utilization (Hojberg et al., 2005) by the gastrointestinal tract (GIT). In consequence, nonutilized nutrients excreted through urine and feces that undergo anaerobic microbial decomposition produce odorous compounds such as volatile amines and sulfurs, phenols, indoles, and volatile fatty acids (Gilley et al., 2000). High concentrations of ammonia and sulfur-containing compounds result in poor performance in broilers (Deaton et al., 1984; Wang et al., 2011), histological changes in the respiratory system, increased susceptibility to disease, and subsequent mortality (Elliott and Collins, 1982; Kristensen and Wathes, 2000). Exposure to high levels of odorous compounds not only adversely affects the health and performance of animals, but can also affect the health of workers and cause environmental problems such as acidification and nitrification of rain (Ferket et al., 2002; Ushida et al., 2003). Accordingly, the Republic of Korea implemented a complete ban of antibiotic growth promoters in animal feed that went into effect in July 2011 (MIFAFF, 2010). Thus, it is necessary to identify new feed additives to ensure the safety of animal products.
Several experiments have reported the beneficial effects of direct-fed microbial (DFM) products provided to broilers on growth performance (Santaso et al., 1999; Mountzouris et al., 2007, 2010), nutrient digestibility (Li et al., 2008; Mountzouris et al., 2010), modulation of intestinal microflora (Koenen et al., 2004; Teo and Tan, 2007; Mountzouris et al., 2010), pathogen inhibition (Mallo et al., 2010; Mountzouris et al., 2010), immunomodulation and gut mucosal immunity (Koenen et al., 2004; Teo and Tan, 2007; Mountzouris et al., 2010), and reduction of ammonia from chicken manure (Endo and Nakano, 1999; Santaso et al., 1999). Bacillus are aerobic, endospore-forming bacteria that have recently shown tremendous promise as DFM candidates because of their survival through the digestive process, germination within the digestive tract, and excretion through fecal matter (Barbosa et al., 2005; Cartman et al., 2008; Shivaramaiah et al., 2011). Bacillus amyloliquefaciens is a potent Bacillus species that produces several extracellular enzymes including α-amylases, cellulase, metalloproteases, and proteases that enhance digestibility and absorption of nutrients in addition to overall immune function of the gut (Gould et al., 1975; Gracia et al., 2003; Lee et al., 2008). Additionally, the bacteriocins (subtilin and barnase) produced by B. amyloliquefaciens (Lisboa et al., 2006; Ulyanova et al., 2011) have bactericidal effects against pathogenic ammonia producing Clostridium perfringensEscherichia coli, and Yersinia. Ferket et al. (2002) found that fecal noxious gas emissions of nonruminants are related to nutrient utilization and the intestinal microflora ecosystem. Experiments with chickens have shown increased nutrient utilization, improved growth performance, increased antibody titers, and balanced cecal microflora in response to being fed a diet supplemented with B. amyloliquefaciens (An et al., 2008). Taken together, these findings indicate that dietary addition of B. amyloliquefaciens may positively improve growth performance and immunity and reduce fecal noxious gas emissions by improving nutrient utilization and intestinal microflora equivalence. Therefore, this study was conducted to assess the effects of supplementation of broiler diets with B. amyloliqufaciens probiotic (BAP) on growth performance, cecal microflora, serum immunoglobulins, and fecal noxious gas emissions from chickens.
Experimental Birds, Design, and Diets
A total of 400 one-day-old male broiler chicks (Ross 308; initial BW 46.1 ± 0.10 g) obtained from a commercial hatchery were weighed and distributed randomly to 1 of 5 dietary treatments in a completely randomized design. Eight replicate pens were assigned to each of the 5 treatments with 10 birds per replicate pen. The pens of each treatment were distributed in such a way that they are located in the front, middle, and back of the house. The dietary treatments were 0, 1, 5, 10, and 20 g/kg of BAP. The probiotic strain used in this experiment was Bacillus amyloliquefaciens KB3, which was provided by the Jeonnam Biodiversity Foundation, Jeonnam, Republic of Korea.
Commercially available broiler diets were used as basal diet prepared with the same batch of ingredients for starter (0 to 21 d) and finisher (22 to 35 d) periods. The ME and CP content of the basal diet was according to the requirements of the Ross-308 rearing guidelines (Aviagen, 2007). The lysine content of the basal diet was somewhat lower (starter: 1.43 vs. 1.18, finisher 1.09 vs. 1.03), whereas the methionine content was higher (starter: 0.51 vs. 0.79, finisher 0.41 vs. 0.70) than the requirement according to Aviagen (2007). The ingredients, chemical composition, and vitamin and mineral content of the experimental basal diets are shown in Table 1. The probiotic product was supplied in powder form and mixed on a weight:weight ratio basis by replacing an equal amount of basal diet. Broilers were kept in a closed, ventilated, wire-floor caged broiler house (100 cm long × 80 cm wide × 40 cm high/cage) at a stoking density of 800 cm2/bird. The cages had a linear feeder in the front and a nipple drinker in the back to provide ad libitum feed intake and free access to water throughout the whole experiment. Temperature was maintained at 33°C for d 1 to 7, after which it was gradually reduced to 24°C at a rate of 3°C per week and then maintained at this temperature until the end of the experiment. The RH was maintained at around 50% throughout the experiment. Continuous lighting was provided throughout the experimental period. All experimental procedures used in this study were approved by the Animal Care and Welfare Committee of the National Institute of Animal Science, Rural Development Administration, Republic of Korea.
Ingredients and chemical composition of the basal diets
Starter (0–21 d) 
Finisher (22–35 d) 
Ingredient (%, as-fed basis) 

Corn grain 
Soybean meal 
Corn gluten 
Soybean oil 
Animal fats 
Dicalcium phosphate 
Vitamin-mineral premix1 
L-Lysine HCl (78%) 
Calculated composition (% of DM unless otherwise specified) 

ME (kcal/kg) 
Crude fat 
Crude ash 
Crude fiber 
Available phosphorus 
1Vitamin-mineral mixture provided the following nutrients per kilogram of diet: vitamin A, 15,000 IU; vitamin D3, 1,500 IU; vitamin E, 20.0 mg; vitamin K3, 0.70 mg; vitamin B12, 0.02 mg; niacin, 22.5 mg; thiamine, 5.0 mg; folic acid, 0.70 mg; pyridoxine, 1.3 mg; riboflavin, 5 mg; pantothenic acid, 25 mg; choline chloride, 175 mg; Mn, 60 mg; Zn, 45 mg; I, 1.25 mg; Se, 0.4 mg; Cu, 10.0 mg; Fe, 72 mg; Co, 2.5 mg (Bayer Korea Ltd., Dongjak-Ku, Seoul, Korea).
Growth Performance
The BW was recorded per pen on a weekly basis from the initial day to the final day of the experiment. In addition, feed consumption for each pen between weighing was determined by measuring feed residue on the same days as the birds were weighed. Feed conversion was calculated as feed per gain based on the weight of feed consumed divided by BW gain per pen. The gain, feed intake, and feed conversion were corrected for dead birds.
Collection and Analyses of Blood Samples
At termination of the feeding trial, 3 birds close to the mean BW were randomly selected from each pen for blood and cecal sample collection. Blood samples were collected (10 mL) from the wing veins of the selected birds into a 10-mL anticoagulant-free vacutainer tube (Greiner Bio-One GmbH, Kremsmunster, Austria). The samples were subsequently stored on ice during the period of collection and then immediately centrifuged to separate the serum (centrifugation for 15 min at 1,610 × g at 4°C). Next, the serum samples were carefully transferred to plastic vials and stored at −20°C until immunoglobulin analysis was performed. The concentrations of serum IgG, IgA, and IgM were assayed using appropriately diluted samples by a sandwich ELISA with chicken-specific IgG (Cat. No. E30–104), IgA (Cat. No. E30–103), and IgM (Cat. No. E10–101) ELISA quantitation kits (Bethyl Laboratories Inc., Montgomery, TX) according to the manufacturer’s instructions. Each experiment was run in duplicate and the results represent the means of triplicate experiments. The absorbance of each well at 450 nm was measured within 30 min using a microplate autoreader (Thermo Lab Systems, Helsinki, Finland). The concentrations of IgG, IgA, and IgM were determined using standard curves constructed from the respective immunoglobulin standards and the results were expressed as micrograms per milliliter of serum.
Collection and Measurement of Cecal Microflora
After blood collection, the selected chickens were exsanguinated by cutting the jugular vein, and the GIT was removed from the carcass. Next, 10-cm segments from the same area of both ceca were dissected and approximately 1 g of cecal content was aseptically collected into a 2-mL safe-lock Eppendorf tube (Thermo Fisher Scientific Inc., Seoul, South Korea) and immediately preserved at −40°C for later microbial analysis.
After thawing, 1 g of the cecal sample was serially diluted with 9 mL of 0.9% sterile saline (1:10 dilution) and thoroughly mixed. Viable counts of bacteria in the cecal samples were then conducted by plating serial 10-fold dilutions in duplicate into MacConkey agar plates, DeMan, Rogosa, Sharpe (MRS) agar plates, and Mannitol Egg Yolk Polymyxin (MYP) agar plates (Difco Laboratories, Becton, Dickinson and Company, Sparks, MD) to isolate the Escherichia coliLactobacillus spp., and Bacillusspp., respectively. The Lactobacillus MRS agar plates were then incubated for 48 h at 37°C under anaerobic conditions, whereas the Bacillus MYP agar plates and E. coliMacConkey agar plates were incubated for 24 at 37°C under aerobic conditions. Microbial colonies were immediately counted after removal from the incubator and expressed as log10 cfu/mL.
Collection of Fecal Sample and Gas Measurements
After finishing the growth trial, fecal samples (mixtures of feces and urine) were collected from the bottom tray of the wire-floor cages of 3 pens per treatment (located in the front, middle and back of the house) into plastic bags and stored immediately at −20°C until use. The total sampled manure from each pen was thawed and then homogenized, after which 500-g subsamples were placed in 2-L plastic boxes in triplicate to measure the NH3 and H2S emissions. Each plastic box was equipped with a cover containing a hole to allow insertion of a gas measuring tube that was sealed inside with adhesive plaster. The samples were allowed to ferment for a period of 3 h at room temperature (24 to 28°C), after which the gas concentration was measured using a Gastec (model AP-20) gas sampling pump (Gastec Corp., Kitagawa, Japan) and Gastec detector tube (No. 3M and 3LA for NH3and 4LT and 4L for H2S). The adhesive plaster was punctured and 100 mL of headspace air was collected from approximately 2.0 cm above the sample surface. After sampling, the tubes were again sealed with adhesive plaster and incubated at room temperature. Additional gas samples were collected at 6, 12, 24, and 48 h. The concentration of NH3 and H2S was expressed as milligrams per kilogram of excreta.
Statistical Analyses
The experiment was carried out as a completely randomized design with 5 treatments. Data were subjected to ANOVA using the PROC IML ORPOL function of the Statistical Analysis System employing polynomial analysis (SAS Institute Inc., 2003). Pens were used as the experimental unit for growth performance parameters [BW, ADG, ADFI, and feed conversion ratio (FCR)] and fecal noxious gas emissions, whereas an individual bird served as the experimental unit for immunoglobulin concentrations and cecal bacterial populations. Orthogonal contrasts were used to determine the linear and quadratic effects of the increasing levels of supplementation of BAP. Variability in the data was expressed as the SE, and a probability level of P ≤ 0.05 was considered statistically significant. Treatment means were computed with the LSMEANS option of the SAS program.
Growth Performance
The effects of dietary BAP supplementation on growth performance traits of broilers at different phases are shown in Table 2. From 0 to 21 d of age, dietary BAP had positive linear effect on ADG (P = 0.003) and negative linear effect on FCR (P < 0.01) of broilers; however, ADFI remained unaffected. From 22 to 35 d of age, dietary BAP exerted positive linear effect on ADG (P < 0.03), with the highest value being observed in response to supplementation with 20 g/kg, whereas ADFI (P > 0.05) and FCR (P = 0.25) were not affected by BAP supplementation. From 0 to 35 d of age, supplementation with BAP had positive linear effects on ADG (P = 0.0005) and ADFI (P < 0.02), and negative linear effect on FCR (P < 0.01) compared with the control, with the highest effect being observed in broilers offered 20 g/kg of BAP.

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